The early and rapid detection of biological toxins is critically important to the protection of security personnel deployed in hostile situations or in instances of domestic terrorism. Biological toxins, such as botulinum toxin, are lethal at very low concentrations, which necessitates detection measures that are both highly specific and extremely sensitive. There are a multitude of scenarios that may require the ability to detect biological toxins at sub-attomolar (10−18M) concentrations or even at levels approaching a few molecules. Foremost, early detection of the use by, for example, terrorists, of biological toxins will allow time for countermeasures, thus decreasing the likelihood of death or injury due to exposure. Civil and military investigative activities require attempts to identify sites of manufacture or storage of biological toxins by soil or water sampling at considerable distances from the suspected site. Such activities may also include attempts to identify former storage sites for biological weapons after the material has been moved and even after attempted sterilization of the site. The military is frequently called upon to clean up storage sites for biological weapons, which requires the ability to survey for residual contamination. Finally, terrorist acts involving biological toxins will frequently require examination of trace forensic evidence for the presence of toxins. All of these examples point to the need for rapid and reliable tests for toxins that are highly specific, but also sufficiently sensitive to detect the target toxin down to the level of a few molecules. In summary, there exists a substantial need for detection capabilities of biological toxins, infectious bacteria and viruses, chemical warfare agents, poisons and other chemical toxins, explosive compounds, and trace forensic evidence.
Presently, cutting-edge techniques employed for the selective high sensitivity detection of protein antigens are antibody-based immunoassays (including “biochip” devices), mass spectrometry, DNA-amplification methods, and nanotechnology techniques. Each of these techniques suffers from drawbacks and problems. Immunoassay methods, such as enzyme-linked immunosorbent assays (ELISAs), employ antibodies directed against a protein antigen to form a highly selective detection method. Assay sensitivity is produced by linking a moiety to the detecting antibody that is capable of some form of signal amplification. However, even under ideal circumstances, ELISA's suffer from detection limits, which are restricted to the nanomolar (nM) to femtomolar (fM) concentration range.
Biochip methods for detecting proteins are a variation of the immunoassay method where antibodies are attached to a membrane in a pattern that can be ready by an optical scanner. The signal amplification methods employed are the same as those for other immunoassays and thus the detection limit is limited to the picomolar level with practical detection limits in the micromolar (10−6M) to nanomolar range (Wang et al., 2001; Cheng et al., 2001; Tanabe et al., 2001; Deng et al., 2001). It is important to appreciate that the greatest advantage of biochip technology is the ability to screen for many antigens at one time (high throughput) rather than high sensitivity for any one antigen.
Nanomolar (10−9M) sensitivity regularly can be achieved with single-shell closed-sphere bilayers (liposomes) with diameters of ˜100 nm containing up to 25,000 fluorescent probes imbedded in each bilayer. Such liposomes can be covalently linked to antibodies and used as the basis of a fluorescence liposome detection method. Since each binding event involves one liposome, signal amplifications of up to 25,000:1 are possible. (Singh et al., 2000).
Greater detection sensitivity can be achieved using amperometric enzyme detection. Enzymes, such as horseradish peroxidase, are linked to the detecting antibody and the product of the enzyme reaction is detected amperometrically through its precipitation on an electrode surface. The amplified electronic transduction of antigen-antibody binding events that results from the activity of the enzyme, forms the basis of the signal amplification (bioelectronic device). This technique permits detection of antigen concentration down to the picomolar (10−12M) level (Alfonta et al., 2001; Doyle et al., 1987). Another technique proving ˜10 femtomolar (10−15M) sensitivity involves the use of fluorescence detection based on highly fluorescent Europium chelates. The chelates are linked to antibodies for detection of antigen-antibody binding. Recent literature reports that heavily labelled Europium chelates polymers (up to 110 total) may be covalently linked to streptavidin based conjugates to detect near femtomolar amounts of prostate-specific antigen (Qin et al., 2001). However, in order to limit background fluorescence to acceptable levels, only small sample volumes (10-20 microliters) can be used for this detection method.
Another approach provides even greater detection sensitivity. When antibodies are coupled to a luminescent protein, such as aequorin from the jellyfish Aequorea victora, the luminescent detection of the bound aequorin yields a detection limit down to the femtomolar (10−15M) level due to the intense bioluminescence of aequorin (Deo and Daunert, 2001; Feltus, et al., 2001). Attomolar (10−18M) concentrations of antigen have been detected with aequorin luminescence, but only by employing a complex optic and detector scheme and by using nanoliter sample volumes (Feltus et al., 2001) to limit the background signal.
Taken as a whole, the immunoassay methods offer outstanding selectivity due to the specificity of the antigen-antibody interaction, but offer only modest sensitivity that is limited in practice to the nanomolar to picomolar concentration range. Higher sensitivities down to the femtomolar range (or the attomolar range for aequorin luminescence) are achievable only with nanoliter sample volumes (in order to limit the protein autofluorescence background signal) and the application of sophisticated optical detection systems that are impractical for field deployable devices.
Advances in mass spectrometric methods, in particular matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, have taken a leading role in the analysis of peptides and proteins (Chaurand et al., 1999). Detection of proteins in biological fluids at sub-femtomolar concentrations is now possible. However, it is not currently possible to uniquely identify a protein, such as a biological toxin, through its mono-ionic species in the MALDI-TOF mass spectrum. As a result, protein identification is achieved by observing the mass spectra of the fragments that result from the proteolytic digestion (typically with trypsin) of the protein and then comparing this spectrum with a library database of known proteins. This process is referred to as protein mapping (Egelhofer et al., 2000). Unfortunately, the kinetics of the proteolytic digestion step limits the lowest practical protein concentration to the micromolar range (Doucette et al., 2000). Recent advances using immobilized tryspin and preproteolytic sample concentration have extended the protein concentration detection limit to the nanomolar level (Doucette et al., 2000). Despite these advances, mass spectrometry clearly lacks both the specificity and sensitivity required for a high sensitivity assay system to detect proteins at the sub-attomolar concentration level.
The most sensitive biological signal amplification scheme ever developed, polymerase chain reaction (PCR), is limited to nucleic acid amplification and cannot directly be used to detect proteins. Indeed, PCR based techniques have already been employed for the high sensitivity detection of organisms that produce biological toxins or disease hazards through PCR amplification of their genomic DNA or RNA (Young et al., 1993). Recently, a new technique combining a hybrid protein assay coupling the use of antibodies directed against proteins and PCR referred to as “immuno-PCR,” has been developed to detect proteins.
Current immuno-PCR techniques employ one of two approaches for coupling amplification substrates (DNA fragments) to antibodies. Direct covalent attachment of the amplification substrate to the antibody of interest is discussed in Wu et al. (2001). This method exploits the terminal phosphate moiety of the amplification substrate, or an amplification substrate modified to contain and amine group, as the basis for the covalent coupling of the amplification substrate to the antibody. Indirect non-covalent attachment of biotinylated amplification substrate and biotinylated antibody to a common streptavidin molecule is described in Sano et al. (1992) and Niemeyer et al. (1997).
In these assays the target protein antigen is immobilized on a substrate (such as a microtiter plate well) and the antibody-DNA complex is allowed to bind to the immobilized antigen. This is followed by the removal of unbound antibody-DNA complex by extensive rinsing. The bound antigen is then detected through the PCR amplification of the amplification substrates attached to the antibody with visualization achieved by gel electrophoresis or a real-time PCR assay. These assays have been employed to achieve detection limits of roughly 6,000,000 (Wu et al., 2001) to 60,000 (Niemeyer et al., 1997) molecules.
Immuno-PCR methods present several difficulties, particularly for field application, as for example, in the military. The detection sensitivity is still relatively low due to extreme potency of some biotoxic agents such as Botulinum or tetanus toxin, detection sensitivity down to the 10-100 molecule level is necessary. The Immuno-PCR methods described above link a single (or at most four) amplification substrates to each antibody. This severely limits the ability of these methods to detect very low copy numbers of antigens (10-100) as detection of only a few copies of the target DNA molecule by PCR is often difficult or impossible. Many samples contain Taq polymerase inhibitors that can inhibit or prevent the replication of low numbers of starting DNA molecules. Furthermore, particularly when in the field, contamination of samples with extraneous DNA is a critical concern for samples with low target DNA concentrations. Finally, even where amplification is successful, it entails a large and time-consuming number of amplification cycles to produce enough DNA to allow for reliable detection of the amplified product.
Recently, three nanotechnology based protein assay methods have been introduced. The first uses atomic force microscopy to perform micro-miniaturized immunoassays on compositionally patterned antibody arrays (Jones et al., 1998). This technique can theoretically detect and identify single antigens based upon the increase in topological height when an antigen binds to an antibody at a specific location in the array. Although highly sensitive and specific, this method requires complex nanofabrication techniques and employs sensitive instrumentation not compatible with use in the field. The other assay methods employ the self-assembly of DNA-streptavidin nanostructures (Niemeyer et al., 1999) or antibody-labeled magnetic beads and sub-micron sized gold particles labelled with antibodies and DNA segments (Nam et al., 2003) for performing immuno-PCR. Although both techniques can detect antigens down to the level of 10-100 molecules, both require sophisticated nanofabrication and detection techniques beyond the capability of most laboratories and researchers.
In summary, none of the current cutting-edge methodologies discussed above are well suited to the development of a highly selective ultra-sensitive assay system for detecting biological toxin hazards in military deployment situations.
Notwithstanding the usefulness of the above-described methods, a need still exists for an ideal assay that would employ antibodies (or other specific receptors) for detection, due to their very high selectivity for proteins, and also employ some form of nucleic acid amplification as a highly sensitive detection method. In order to extend the detection limit down to 10-100 molecules of antigen a method is required to overcome the limitations associated with low initial copy numbers of amplification substrates that severely limit the current immuno-PCR methods. This is achieved in our ILNAA assay method by attaching antibodies (or other specific receptors) to closed-shell liposomes of about 100 nm in diameter that encapsulate 50-1000 amplification substrates inside each liposome. In this way each antibody-binding event is associated with 50-1000 amplification substrates rather than one amplification substrate as is the case for the current immuno-PCR methods. This approach overcomes the difficulty in initiating the amplification reaction starting with just a few amplification substrates. For example, for 10 bound antigens there would be only 10 amplification substrates with which to initiate the PCR reaction with the current immuno-PCR methods, but 500 to 10,000 with our ILNAA assay method. This improvement in the number of amplification substrates per binding event also serves to proportionally reduce the problems associated with background DNA or RNA contamination, which improves proportionally with the initial number of amplification substrates present at the start of PCR or RT-PCR. Finally, our ILNAA assay method presents a unique approach for further reducing the background nucleic acid contamination. In our assay the amplification substrates are encapsulated inside closed-shell liposomes and, as such, are sequestered from the rest of the assay solution.
This allows for the addition of DNase or RNase to the assay solution as a means of degrading any background DNA or RNA present that could be amplified by the nucleic acid amplification step and present a false positive, reducing the sensitivity and reproducibility of the assay. The enzyme can be heat deactivated prior to the rupture of the liposomes so that the released amplification substrates are the only source of nucleic acid capable of being amplified. In the current immuno-PCR methods the amplification substrates are exposed to the bulk assay solution making DNase reduction of background DNA impossible as the amplification substrates themselves would also be degraded. For the above reasons our ILNAA assay method represents a significant enhancement over existing high-sensitivity assay systems and solves the problems associated with the highly selective detection of antigens at extremely low copy numbers.